SPECTROSCOPY
Mr. Nasir Ali
Asst. Prof.
Lahore Pharmacy College
CONTENTS
Electromagnetic spectra
Introduction to spectroscopy
Ultraviolet and Visible Spectroscopy
3
ELECTROMAGNETIC
WAVES
Electromagnetic (EM) waves are transverse waves. Electric and magnetic fields
oscillate perpendicularly to create electromagnetic waves (EM waves), which do
not require particles to do so. Energy is transferred from a source to an absorber
through EM waves.
Where,
E = Direction of electrical field
B = Direction of magnetic field
V = Direction of wave velocity
ELECTROMAGNETIC SPECTRA
4
Wavelength increases
Wavelength is inversely
proportional to energy
Note there are
total seven
spectra
5
DESCRIBING AN EM RADIATION
WAVELENGTH WAVENUMBER FREQUENCY ENERGY FORMULA
f E
Number of waves • Different EM
• Distance between •
Number of waves passing through a radiations have ℎ
2 consecutive •
crusts or troughs per unit length point per second different energies E=
depending upon 𝜆
• Units: µm, nm, • Units: cm-1 • Units: Hertz (Hz),
wavelength
pm Cycles sec-1 • Unit: Joule (J), kJ
6
MATTER-RADIATION INTERACTION
ELECTRONIC TRANSITIONS CHANGES IN MOLECULAR RESONANCE WITH
VIBRATIONS SPINNING NUCLEUS
• Electrons are excited from • Chemical bonds having • Different nuclei having odd
low energy orbit to high some dipole moment atomic number or odd
energy orbit by absorbing vibrate at specific frequency mass number or both show
radiations • Absorption of specific nuclear spin
• Excited state electrons come radiations causes the • Nuclear spin results into
back to low energy orbit by change in these vibrations generation of magnetic
releasing radiations • Absorbed and transmitted field
waves can be determined • Resonance of applied EM
rays with nucleur spin
ULTRAVIOLET AND
VISIBLE
SPECTROSCOPY
8
UV/VIS SPECTROSCOPY
ELECTRONIC
UV-Range VIS-Range ABSORPTION TRANSMITTANCE
TRANSITIONS
190 – 400 nm 401 – 800 nm 4 important types Beer’s Lambert Law Inverse of absorbance
9
INSTRUMENTATION
SINGLE AND DOUBLE BEAM 10
UV/VIS SPECTROPHOTOMETERS
11
DOUBLE BEAM UV/VIS
SPECTROPHOTOMETER
HYDROGEN DISCHARGE 13
LAMP-SOURCE OF UV LIGHT
The electrical
excitation of
deuterium or
hydrogen at low Both deuterium and
hydrogen lamps emit
pressure produces
radiation in the range
a continuous UV 160 - 375 nm.
light. (deuterium discharge
lamp 185 – 380 nm)
Deuterium lamps
give approximately
five times greater
light intensity than
hydrogen lamp
Quartz windows must when operated at
be used in these lamps, same wattage.
and quartz cuvettes
must be used, because
glass absorbs radiation
of wavelengths less
than 350 nm.
TUNGSTEN LAMP-SOURCE 14
OF VIS LIGHT
•The bulb contains a filament of Tungsten fixed in evacuated
condition and then filled with inert gas
•The filament can be heated up to 3000 k, beyond this Tungsten starts
sublimating
• To prevent this along with inert gas some amount of halogen is
introduced (usually Iodine).
• The halogen prevents evaporation of the tungsten and increase life of
lamp
Sublimated form of tungsten reacts with Iodine to form Tungsten –
Iodine complex which migrates back to the hot filament where it
decomposes and Tungsten get deposited
MONOCHROMATOR
15
By rotating prism or grating
we can get the light of
desired wavelength.
16
SAMPLE HOLDER 17
the cells or
cleaning;
cuvettes are used
washing with
for handling
distilled water or
liquid samples.
with dilute
alcohol, acetone
surfaces of
absorption cells
must be kept rectangular or
clean; no cylindrical cells
fingerprints or
blotches
visible region; for study in uv
color corrected region; quartz
fused glass cells cells
DETECTORS
18
Device which converts The following types of
light energy into detectors are employed in
electrical signals, that are instrumentation of
displayed on readout absorption
devices. spectrophotometers
The transmitted radiation
falls on the detector
which determines the
intensity of radiation
absorbed by sample
BARRIER LAYER CELL
PHOTOTUBE
PHOTOMULTIPLIER
READ OUT DEVICE
THEORETICAL
ASPECTS
24
BEER LAMBERT’S LAW
The absorbance of a solution is linearly proportional to its
concentration and path length of light in solution”
A = ε cl
Where,
A = Absorbance of sample or optical density or extinction co-efficient = log Io / I
ε = molar absorptivity (liter M-1cm-1)
l = length of the light path through the sample, in centimeters
c = concentration of the sample, in moles/liter
I = intensity of the radiation emerging from the sample
Io = intensity of the radiation entering the sample
25
MOLAR ABSORPTIVITY (ε)
The molar absorptivity (ε)
of a compound is a
constant that is
characteristic of the
compound at a particular
wavelength.
The solvent in which the
(The abbreviation comes
sample is dissolved is
from the fact that molar
reported because molar
absorptivity was
absorptivity is not
formerly called the
exactly the same in all
extinction coefficient.)
solvents.
The molar absorptivity
It is the absorbance that
of acetone dissolved in
would be observed for a
hexane, for example, is
1.00 M solution in a cell
9000M-1cm-1 at 195 nm
with a 1.00-cm path
and 14 M-1cm-1at 274
length.
nm.
26
SPECIFIC ABSORBANCE (A1%, 1cm)
Another form of the Beer-
The most common form in The beer- Lambert
Lambert proportionality
Pharmaceutical analysis is equation therefore takes
constant is the Specific
A (1%, 1cm), which is the the form
absorbance, which is
absorbance of 1g/100ml
absorbance of a specified A=A 1% c l
(1%w/v) solution in a 1 cm
concentration in a cell of
cell.
specified pathlength.
27
LIMITATIONS OF BEER-LAMBERT LAW
Under ideal conditions However, under real situations a
absorbance versus curvature in the plot is observed
concentration plot is a straight beyond a particular
line passing through the concentration.
origin.
CHROMOPHORE 28
Nitro group
gives yellow
any group which color to
compound
exhibit absorption
of electromagnetic
radiation in a visible
or ultra-visible
Previously used
region
for colored part
of a molecule
It may or may
not impart
any color to
the compound
CHROMOPHORE 29
CHROMOPHORE AND AUXOCHROMES
30
CHROMOPHORE AND AUXOCHROMES
31
CHROMOPHORE AND AUXOCHROMES
32
UV SPECTRUM
33
UV SPECTRUM
34
SHIFTS IN LAMDA MAX
35
Absorbance
36
ELECTRONIC TRANSITIONS
37
ELECTRONIC TRANSITIONS
σ-σ* n-σ*
• Observed in saturated • Observed in saturated
compounds compounds with
• Large energy required heteroatoms (N, O, S, X etc.)
• Absorption occurs in vaccum • Relatively less energy
UV region (below 190 nm) required
• e.g in CH4 (125 nm) • Absorption occurs towards
shorter wavelength of UV
region (150-250 nm)
• e.g in CH3OH (227 nm)
38
ELECTRONIC TRANSITIONS
π-π* n-π*
• Observed in unsaturated • Observed in compounds
compounds (double, triple containing double or triple bonds
bonds or aromatic ring) like involving hetero atoms (C=O,
alkenes, alkynes, carbonyls, N=O)
nitriles, aromatics etc • Requires minimum energy
• Relatively less energy required
• Absorption occurs towards
• Absorption occurs UV region loingeer wavelength of UV
• e.g in 1,3-Butadiene (217 nm) region
• Conjugation increases the • e.g in Aldehydes (290 nm)
wavelength
APPLICATIONS 39
Qualitative
determination
Structural Quantitative
analysis determination
Quality
control (QC)
QUALITATIVE
ANALYSIS
SCAN Adjustable scan range
200-800 nm
Determination of lamda max and
comparing with standard
UV SCAN OF IBUPROFEN
240-300 NM
SCAN Lamda max at 264 nm and 272 nm
Peaks show a mix of saturation and
unsaturation
UV SCAN OF IBUPROFEN
240-300 NM
264 nm
272 nm
UV SCAN OF IBUPROFEN
COMPARISON WITH
STANDARD
QUANTITATIVE
ANALYSIS
Specific
absorbance
Single standard
Multiple
standard
SPECIFIC ABSORBANCE METHOD
45
Making suitable Scaning to
Taking
dilutions establish lamda
Absorbnace absorbance
max
less than 1
46
47
THANKS 48
